1. Field of the Invention
The invention relates generally to protease assays.
2. Background Information
Proteases play a vital role in the viability and regulation of cellular activity. Proteases act inter- and intramolecularly, to activate and inactivate proteins, and regulate expression of proteins by their action with transcription factors and transcription factor regulating proteins. Proteases are active in blood clotting and embolism dissolution, apoptosis, inflammatory activity, processing of proteins, metabolism, degradation of proteins, etc. The processes are greatly varied as to their action, mechanism and function. Proteases come within the class of hydrolases, hydrolyzing amide bonds. For this purpose, there are numerous classes of proteins, such as the serine/threonine hydrolases, metalloproteinases, cysteine proteases, etc. While many proteases are promiscuous in their recognition sequences, such as trypsin, chymotrypsin, bromelain, papain, etc., having fairly common recognition sites, many other proteases have recognition sequences that are rare except for the particular protease substrate.
In addition, there are many microorganisms that depend upon specific protease activity for their infectivity. Being able to inhibit proteases essential to the viability of the organism would diminish the infectivity of the organism. Viruses depend to a great degree on expressed proproteins that are cleaved to active products. Inhibiting such selective cleavage would inhibit the viability of the virus. There is, therefore, an interest in providing methods that can detect the presence of a specific protease in a sample, be capable of being used for rapid screening, be sensitive to the particular protease at low concentrations of the protease, while being reasonably stable to other proteases, and provide for a ready reliable readout.
Recently, in WO 00/39348 and references cited therein, a system is described that employs α-complementation between a small fragment of β-galactosidase called the enzyme donor fragment (“ED”) and a larger fragment referred to as the enzyme acceptor (“EA”), where the two fragments complex to form an active β-galactosidase. The method described in the aforementioned application fuses the ED to a protein of interest, where there is a recognition sequence in the protein of interest. The fusion protein is reported to have substantially less activity than the protease catalyzed product. This method has numerous deficiencies. One of the advantages of the ED use in the appropriate environment is that it is readily degraded intracellularly, so that ED, by itself, does not provide a background. Where the ED is cleaved from the protein of interest, it may be rapidly degraded, so as to confuse the result. Furthermore, the inhibition of complexing of ED to EA is difficulty achieved, so that the fusion protein will have significant activity. Since initially the fusion protein will be present in much greater amount than the cleavage product, one will be dealing with small differences in observed signal, substantially reducing the sensitivity of the assay.
One of the issues associated with assays is the preparation of reagents in pure form. Frequently, purification can be difficult since impurities can lead to background interference with the assay, inhibitors of enzymes or other interfering aspects. In addition, purification can substantially add to costs, where there is reduction in the amount of product, the cost of the purification protocol and the requirement for quality analysis. There is great interest in devising ways to reduce the cost of reagents and avoid purification and quality control.